Toxicity of chemicals to organisms is important in determining how chemicals in an environmental sample will behave in nature. Various types of tests have been used to determine the extent of toxicity of chemicals to microorganisms. Microbial toxicity tests are advantageous because of the short life cycles of the organisms and the high test concentrations which can be used.
Liu, "A Rapid Biochemical Test For Measuring Chemical Toxicity", Bull, Environm. Contam. Toxicol., 26, 14-149 (1981), incorporated herein by reference, describes a biochemical toxicity test based on reduction of resazurin by bacterial respiration. Resazurin is a redox-active dye which, when reduced, changes color from blue to pink. Substances which are toxic to bacteria can inhibit their metabolism and thus inhibit the rate of resazurin reduction. The method as described by Liu involved an organic solvent extraction and centrifugation. These steps are time consuming and cumbersome. Also, the extracting solvent used, n-amyl alcohol, has toxicity problems associated with it.
A commercially-available test known as "MicroTox" measures the effect of toxins on the light output of luminescent bacteria. Bioluminescence is not an essential metabolic process nor is it widespread among living organisms. Toxins that specifically inhibit luciferase may not exhibit general toxity to other organisms. In addition, measuring luminescence requires use of a very expensive instrument.
Another commercially-available test is known as "Toxi-Chromotest" which measures the cleavage of X-Gal or ONPG by the .beta.-galactosidase enzyme of a strain of Escherichia Coli that is highly sensitive to toxins. What is actually measured in this test is the ability of the organism to synthesize the enzyme. The test is limited to the use of one genetically engineered strain of E. coli.
Another commercial test is known as the IQ Toxicity Test which measures the cleavage of a fluorescent dye by the .beta.-glucuronidase enzyme of Daphnia (water fleas). In this case the activity of one enzyme is used to represent the viability of the organism.
The PolyTox test is also commercially available. It measures the reduction of oxygen consumption by bacteria. It requires the use of a dissolved oxygen electrode. Only one test can be run at a time. The information gained from this test is similar to that obtained from the resazurin reduction method but it requires more expensive equipment, and multiple tests require more time and sample manipulation.
The foregoing tests are all designated to be used with a specific organism. Users are not given the option of determining the toxicity of substances to their own biomass.
There has not heretofore been described a simple and rapid chemical toxicity test of the type described herein.
Respiration is the process that generates ATP (adenosine triphosphate) using the energy from the oxidation of an electron donor by an external electron acceptor. In the case of aerobic respiration the terminal electron acceptor is O.sub.2. Electrons are transferred from the electron donor to the terminal electron acceptor through a series of electron-transfer proteins that are imbedded in the cell membrane. Some substances, such as redox-active dyes like resazurin, can act as alternative electron acceptors by oxidizing one of the membrane proteins.
Electron transfer is coupled to ATP generation by proton gradient across the cell membrane that is created as electrons flow through the membrane-bound electron transfer system. ATP is synthesized from ADP (adenosine diphosphate) and inorganic phosphate when this proton gradient is used to drive the phosphorylation of ADP by a membrane-spanning ATPase.
Since it involves the creation and maintenance of a proton gradient across the cell membrane, respiration is affected by substances that make the membrane permeable to protons. When protons can cross the membrane freely, bacteria can not use a proton gradient to make ATP. The oxidation of energy sources is thereby uncoupled from the phosphorylation of ADP. Substances that uncouple oxidation from phosphorylation in this way are often organic acids like phenols that are lipid soluble but can become protonated on the outside of the membrane, diffuse across the membrane and then become deprotonated on the inside of the membrane.
Since the rate of catabolic processes is controlled by the ADP/ATP ratio, these processes are accelerated by the uncoupling of oxidation from phosphorylation. Some substances that cannot act as proton shuttles uncouple oxidation from phosphorylation by inhibiting the ATPase that uses the proton gradient to synthesize ATP.
The result of the action of uncouplers in toxicity test is twofold. Reactions that are dependent upon the concentration of reduced coenzymes and ATP are slowed due to a decrease in the concentration of substrates. Reactions like the reduction of resazurin that are dependent upon the rate of electron transfer are accelerated.